Molecular Diagnostics Flashcards
Requirement for PCR and hybridization
the genome of the pathogen and humans is known
single stranded DNA binds to another strand of DNA or RNA; useful to detect and quantify the target DNA or RNA;
Hybridization (blotting)
Northern Blotting
DNA probe and mRNA is target; gene expression
Southern Blotting
Both probe and target are ssDNA; looks at restriction fragments
Western Blotting
Proteins; determines Ab or protein present
Eastern Blotting
Identifies PTMs on proteins
PCR steps
Denature, anneal, extend
3 requirements of PCR
primers, dNTPs, and Taq polymerase
Quantitative PCR
PCR that includes a probe that fluoresces only in the presence of the PCR product; levels of an infectious agent or levels of gene expressed
Restriction Fragment Length Polymorphism (RFLP)
differences in restriction site sequences of DNA found when restriction endonucleases added and the fragments placed on a gel; forensics, paternity testing, and disease detection
How to test for sickle cell
RFLP; normally 3 restriction sites in beta-globulin but only 2 in sickle cell individuals
Variable Number of Tandem Repeats
isolated by flanking restriction sites or via PCR; key for several inherited diseases
cDNA of the protein is inserted into expression vectors
Recombinant proteins
Normal insulin
proline at 28, lysine at 29 at C terminus of B chain
Lispro
switches proline and lysine
